Novel Assays for Measuring Susceptibility to Inhibitors that Target HIV1 Virion A

描述（由申请方提供）：本项目的总体目标是开发新型、稳健、高通量检测方法，以评价对HIV-1 Gag和蛋白酶蛋白抑制剂的敏感性，特别强调病毒体组装和成熟。这些检测将基于Monogram Bioscience广泛使用的PhenoSense HIVTM和GeneSeq HIVTM耐药性检测的成熟技术，即首先开发的基于重组病毒和基于核酸测序的检测，以评估HIV-1蛋白酶和逆转录酶的抑制剂。目前，几种组装/成熟抑制剂正在临床前和临床研究中进行评估，例如bevirimat（Panacos）、vivecon（Myriad Genetics）。因此，需要可靠的高通量检测来支持这种有前途的新型抗逆转录病毒药物的开发工作，并最终将其用于常规临床实践。自2000年在Monogram临床参考实验室实施并商业化以来，PhenoSense HIV和GeneSeq HIV检测试剂盒已用于表征超过150，000份患者样本。此外，在过去的八年中，这两种检测方法已被广泛用于支持抗逆转录病毒候选药物的III期评价，以表征对新药的耐药性并指导优化背景方案的选择。2003年，Monogram成功地采用了PhenoSense和GeneSeq技术来评估HIV-1进入抑制剂（例如恩夫韦肽、马拉韦罗、vivcriviroc、ibalizumab、PRO-140），今年我们完成了两种新检测方法的验证，以监测对整合酶抑制剂（例如雷特格韦、埃替格韦）的耐药性。该申请描述了一种简单、可靠的策略，用于开发PhenoSense和GeneSeq HIV耐药性检测系统，该系统能够评估HIV-1 Gag和蛋白酶蛋白的抑制剂;特别是靶向病毒组装和成熟的抑制剂。由于这些新的检测系统将包括免疫优势Gag区，它们很可能被证明对细胞免疫反应的研究有价值。这些研究可能对疫苗的设计和评价有价值。公共卫生相关性：成功完成资助提案将导致新的基因型和表型检测的开发和验证，这将使耐HIV-1组装和成熟抑制剂的病毒的特性，并将有助于指导HIV-1感染患者的抗逆转录病毒治疗决策。该试验还将大大推进gag特异性CTL逃逸变体的基因型和表型表征，并将证明对改进的HIV疫苗和免疫调节疗法的设计和开发有价值。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop novel, robust, high-throughput assays to evaluate the susceptibility of to inhibitors of HIV-1 Gag and protease proteins, with particular emphasis on virion assembly and maturation. These assays will be based on proven technologies underlying Monogram Bioscience's widely used PhenoSense HIVTM and GeneSeq HIVTM drug resistance tests, i.e. recombinant virus-based and nucleic acid sequencing-based assays first developed to evaluate inhibitors of HIV-1 protease and reverse transcriptase. Currently, several assembly/maturation inhibitors are undergoing evaluation in pre-clinical and clinical studies, e.g. bevirimat (Panacos), vivecon (Myriad Genetics). Consequently, reliable, high-throughput assays are needed to support the development efforts of this promising new class of antiretroviral drugs, and eventually, their use in routine clinical practice. Since their implementation in the Monogram Clinical Reference Laboratory and commercialization in 2000, the PhenoSense HIV and GeneSeq HIV assays have been used to characterize in excess of 150,000 patient samples. In addition, both assays have been used extensively to support phase III evaluations of antiretroviral drug candidates over the past eight years, both to characterize resistance to new agents and to guide the selection of optimized background regimens. In 2003, Monogram successfully adapted the PhenoSense and GeneSeq technologies to evaluate inhibitors of HIV-1 entry (e.g. enfuvirtide, maraviroc, vivcriviroc, ibalizumab, PRO-140), and this year we completed validations for two new assays to monitor resistance to integrase inhibitors (e.g. raltegravir, elvitegravir). This application describes a straightforward, tried and true strategy to develop PhenoSense and GeneSeq HIV drug resistance assay systems that are capable of evaluating inhibitors of HIV-1 Gag and protease proteins; specifically inhibitors that target virus assembly and maturation. Because these new assay systems will include the immuno- dominant Gag region, they arelikely to prove valuable to the study of cellular immune responses. Such studies may prove valuable to vaccine design and evaluation. PUBLIC HEALTH RELEVANCE: Successful completion of the funding proposal will result in the development and validation of new genotypic and phenotypic assays that will enable the characterization of viruses that are resistant to HIV-1 assembly and maturation inhibitors and will help guide antiretroviral treatment decisions for HIV-1 infected patients. The assay will also greatly advance the genotypic and phenotypic characterization of gag-specific CTL escape variants and will prove valuable to the design and development of improved HIV vaccines and immune-modulating therapies.